1. Field of the Invention
The present invention relates generally to reagents for determining kinase and phosphatase activity, and more specifically to chimeric proteins containing two fluorescent proteins and a phosphorylatable domain, and methods of using such chimeric proteins to detect kinase or phosphatase activity.
2. Background Information
Phosphorylation is the most important way that individual proteins are post-translationally modified to modulate their function, while practically all signal transduction involves dynamics of protein-protein interaction. Various technologies have been used to enumerate the main phosphorylation/dephosphorylation events and interacting protein partners involved in cell function, including, for example, function of cardiomyocytes and B lymphocytes. However, the most common currently used technologies such as two dimensional gel electrophoresis, mass spectrometry, co-immunoprecipitation assays, and two-hybrid screens require destroying large numbers of the cells or transferring genes to heterologous organisms. As such, these methods have poor temporal and spatial resolution, and are insufficient to directly probe physiological functions such as contracture or chemotaxis, which occur on the time scale of milliseconds to minutes.
The most widely used method for detecting phosphorylation of specific proteins in single cells utilizes antibodies that discriminate between the phosphorylated and dephosphorylated forms of an antigen. Such antibodies can, in principle, reveal the phosphorylation state of the endogenous protein just prior to the time the cells were fixed for examination, without any introduction of exogenous substrates. However, the identification of antibodies that can discriminate between a phosphorylated and unphosphorylated form of a protein is time consuming and expensive. In addition, the necessary immunocytochemistry is tedious, and is difficult to reassemble into a quantitative time course.
In order to achieve dynamic recording of phosphorylation in single cells, peptides have been labeled with acrylodan, a probe whose fluorescence can be sensitive to the phosphorylation of the peptide. For example, when acrylodan was attached to a peptide from myosin light chain, an approximately 40% decrease in emission peak amplitude upon phosphorylation in vitro was observed. When microinjected into fibroblasts, the peptide incorporated into stress fibers, but no dynamic changes were observable. Substrates for CaMKII and PKA also have been labeled with acrylodan and, after exposure to the kinase, fluorescence was about 200% and 97%, respectively, of initial values. These peptides were hydrophobic enough to stain live cells, and local intensity changes of up to 10% to 20% of initial fluorescence were seen in some regions. The fluorescence of the PKA substrate simultaneously decreased in the cytosol and increased in the nucleus by an amount that was greater than could be explained by the in vitro sensitivity, indicating that more complex factors such as translocation were dominating.
Although no follow-up studies on the use of these acrylodan-labeled peptides have been reported, the approach of developing phosphorylation-sensitive fluorescent substrates may be worth pursuing. However, the use of acrylodan-labeled peptides provides no rational mechanism for phosphorylation sensitivity. Thus, a need exists for phosphorylation-sensitive indicators that can be used to detect phosphorylation or dephosphorylation events in a cell. The present invention satisfies this need and provides additional advantages.